As a student, postdoctoral fellow and instructor, I have studied the molecular biology and signal transduction mechanisms of G protein coupled receptors (GPCRs). Over the past 4 years, I investigated the neurobiology of estrogen action in the control of female reproductive function and in neuroprotection following brain insult. My experience in these two fields stimulated my interest in understanding the molecular basis of sex differences in and hormonal control of addictive disorders and psychiatric diseases. Indeed, sex differences in drug abuse liability are well documented, and there is growing evidence that estrogens potentiate drug reinforcement. My recent observation that the newly discovered estrogen receptor GPR30, which is a GPCR, is specifically expressed in dopamine cells of the ventral tegmental area (VTA) that project to the nucleus accumbens prompted me to examine the possibility that this GPCR plays a role in the hormonal regulation of drug addiction. The study of drug reinforcement and the neurobiology of addiction is an entirely new orientation for my career, and the topic to which I will devote my scientific career. During the mentored award period, I will learn several new methods needed to support my development as a drug abuse researcher. I will receive training from Dr. Mary Kritzer, a highly skilled neuroanatomist who has made seminal observations on the expression of sex steroid receptors in brain regions involved in motivated behaviors, and Dr. Mark Wightman, who is world renowned for the development of methods for real time measurement of synaptic dopamine levels by fast scan cyclic voltammetry in awake animals. I will be assisted by my primary mentor Dr. Saleem Nicola to properly design and carry out cocaine self administration experiments. My co- mentor, Dr. Anne Etgen will continue to advise me on mechanisms of estrogen action in the brain. This will equip me to develop independent research projects on the role of sex steroid hormones in the brain that do not overlap with the research projects going on in the lab of either of my mentors. This training will take place at an institutional environment with outstanding facilities and a long history of collaborative interactions among the research faculty. These experiences will facilitate achievement of my long term career objective, to conduct research in my own laboratory as a faculty member at a university or academic medical center. My long-term research goal is to determine the molecular basis and neural circuits that underlie sex differences in drug abuse liability using a combination of behavioral, electrochemical, molecular and pharmacological techniques. The proposed research plan tests the hypothesis that estradiol ( E2) enhances cocaine reinforcement by elevating synaptic dopamine levels in the nucleus accumbens, and that GPR30 mediates these actions of E2. Clinical and preclinical evidence indicates that ovarian steroid hormones, particularly estrogens, modulate dopamine neurotransmission, and that this may be relevant to sex differences in drug reinforcement, with females being more vulnerable than males to drug addiction and relapse. It is well documented that estradiol and related estrogens have rapid actions in the brain that can be observed within seconds to minutes, suggesting that estrogen binding molecules expressed at the plasma membrane participate in mediating cellular responses to estradiol. GPR30, a GPCR which was recently shown to mediate estradiol activation of several cell signaling pathways in vitro, is a potential candidate for mediating rapid estradiol regulation of brain functions. My preliminary immunohistochemistry studies show that GPR30 is highly expressed in midbrain dopamine neurons. Surprisingly little is known about the influence of ovarian steroids on the mesolimbic drug reward circuits, especially the possibility that estradiol rapidly modulates synaptic dopamine availability in the nucleus accumbens. Therefore, the proposed specific aims test the hypothesis that estradiol acts via GPR30 to enhance cocaine reinforcement by elevating synaptic dopamine levels in the nucleus accumbens. The proposed studies will employ a combination of immunohistochemistry and tract tracing, in vivo administration of estradiol or a specific agonist for GPR30 (G1) and in vivo knockdown of GPR30 in the VTA to investigate the role of GPR30 in mediating estrogen regulation of cocaine reinforcement and dopamine release in the nucleus accumbens. Aim 1 will use double-label immunocytochemistry for GPR30 and tyrosine hydroxylase, a marker of dopamine neurons, combined with retrograde tract tracing to identify the neuroanatomical distribution of GPR30-expressing neurons in the cortico-mesolimbic dopamine system. Aim 2 will determine whether the GPR30 agonist G1 mimics the ability of estradiol to potentiate cocaine reinforcement as measured by facilitation of the acquisition of cocaine self administration and to increase motivation to take cocaine under a progressive ratio schedule. We will then assess E2/G1 modulation of cocaine reinforcement in animals subjected to GPR30 knockdown using in vivo RNA interference targeted to the VTA. Aim 3 will employ a combination of in vivo infusions of E2 and G1, fast scan cyclic voltammetry measurement of dopamine release in the nucleus accumbens and in vivo RNA interference to knock down GPR30 in the VTA to test the hypothesis that activation of GPR30 modulates both tonic and phasic dopamine release specifically in the nucleus accumbens shell in response to cocaine. These studies will provide rigorous training for my future research in drug addiction. They will also provide insight into the general molecular mechanisms underlying presynaptic regulation of dopamine neurotransmission in the nucleus accumbens by estradiol and thus may also shed light on the basis of sex differences in drug abuse liability. PUBLIC HEALTH RELEVANCE: There is compelling evidence that women are more vulnerable than men to many different aspects of drug addiction. The proposed research will examine the neurobiological and molecular basis of the regulation of drug reinforcement by a major female sex steroid hormone, estradiol.